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1956 (IX)

[00:00:00] This is Roman book, physical Roman IX. Starts 1953, August 29th, and runs through October 29th or thereabouts. Very straightforward beginning. Just trying some gels with — for cholinesterase. Almost certainly this was encouraged by Dave Poulik but I’m not certain at this point. But later on he became important in this work. He was my friend and colleague. So freshly thawed serum. Cholinesterase [00:01:00] came out well with two faint leading zones which faded overnight. It gives the formula for the stain. The substrate was betacarbonaphthoxycholine iodide. So beginning to do enzymograms. Straight full family on August 29th. Pages are not numbered in this book. Two-dimensional gels August 30th. Good stain for the enzyme and overstain with amido black for two-dimensional tests. Good stain with bromophenol blue on top of the enzyme. Some photography of the samples. [00:02:00] Gel was run on August 20th or at least recorded by Otto. Thursday, Friday, August 31st, some two-dimensional — and a two-dimensional experiment. Looking at cholinesterase as well. There is a rather good account of these electrophoresis experiments that I’ve already mentioned published by me around about 2010 or thereabouts. A history of starch — of gel electrophoresis. It’s worth recording. [00:03:00] Beginning to try some other stains. Friday August 31st trying to stain with oil red O to stain for lipoproteins of lipids. Recheck of starches August 31st. Some starches. A lot of starch, 541117 was an old lot of starch. And some comment on staining with oil red O. Lipoprotein photography mating. [00:04:00] Trying to determine on Monday September 3rd if the lipoproteins are improved if a slit is used instead of filter paper for the inserting the sample. And this is clearly the post slow alpha 2 region is better with a spacer, etc., etc. Two-dimensional gels again. Tuesday September 4th. Trying all sorts of solutions of [00:05:00] oil red O in different solvents. Not realizing that the actual staining ability is constant if there is solid material there. In the saturated solution. I have the note. Theoretically unsound experiment as all solutions are saturated in oil red O and so have equal activities. That’s activity being in the thermodynamic sense. And therefore equal staining powers. And they’re almost — but using 90% methanol certainly dissolves the lipids. More gels run by Otto. [00:06:00] On September 5th. Comment on the summary of slit experiment and on oil red O experiments. Nothing very striking. More samples on September 6th. And a crazy. Zone electrophoresis in a zigzag system. Tried shortly after September 6th. I don’t have a particular date. Aim to stabilize against density gradient by using a pathway system. But it was quite crazy and didn’t work. There were one, two, three pages of trying to make it work. [00:07:00] Multiple myeloma sample September 7th heavy haptoglobin. Juvenile diabetic family on Saturday September 8th with Otto’s writing. Monday September 10th gel again with Otto’s writing. September 12th repeated the arch chip in family. And then some ideas of trying to do the first dimension for [00:08:00] two dimensions in a starch grain type electrophoresis of the sort used by Kunkel. And then blotting the gel after. Blotting the starch grains afterwards. So the idea was to build up the starch and then blot it dry and run the sample and then blot out onto filter paper, instead of using filter paper electrophoresis I’d use starch grain electrophoresis and blot it onto filter paper. And I was determining how much starch to use, etc. With a diagram of the starch result. Bridge solution, etc. This is about September 13th, Thursday, September 13th. [00:09:00] This says starch result. Separation fairly good by the morning. But a blue streak centrally on top, etc. Not well packed on the top. Better have closed system, etc. So then this is — I’m concerned with electroosmosis on the filter paper or in the starch. The results show no significant difference in the migration but at 45 minutes there was a difference, etc., etc. Leakage and till electroosmosis and not very significant experiments. You can — the filter paper, one can see [00:10:00] tests of what was going on. Just about September 13th. Trying to improve the two-dimensional. Beginning to look at some samples from cattle eventually. Test system and control bovine hemoglobin. Just a test of the gel system. Why I was trying with bovine hemoglobin. Several pages of working on [00:11:00] electroosmosis. Saturday September 15th. Some more family studies. Beginning on September 18th Otto’s entries, notations. Otto’s gel. Juvenile diabetic family. September 21st again with Otto’s. All this is Otto’s writing. And one sample here from a person who had severe multiple transfusion reaction. Pyrogenic reactions. Run on the 21st to see if one could see anything special about it. Was a person with myeloid leukemia question mark [00:12:00] that had had several occasions previously a transfusion reaction. But there’s no comment on anything particularly unusual that was found. More samples with Otto. Going for the next several pages here. Tuesday September 26th. And then mail on 25th of September. Received on Wednesday 26th some Negro samples as I call them. We now call them African American. From New York. Those were from Jim Engels and Ralph. I’ll get his name. [00:13:00] In a moment. On Wednesday September 26th I record receiving Negro samples from New York that were mailed on the 25th. They were sent to me by two investigators, Ralph E. Engle, Jr. and James H. Pert at New York. I don’t remember which hospital in New York. Yes. Ralph Engle and Jim Pert were at New York Hospital in New York. And this is the beginning of our discovery of inherited [00:14:00] differences in the transferrins. Plasma protein transferrin. Which at that time I just called beta-globin. I didn’t know that it was — didn’t yet know that it was a transferrin. To me there’s a little sadness about this story because I did publish eventually this variation in the sample. And I thanked Ralph Engle and Jim Pert for sending me the samples. But really they ought to have been authors on the paper and I haven’t the faintest idea why I didn’t think of it at the time. It was not a selfish act. It was an act of just forgetting that it would have been much nicer to have them as authors. I often think of this as an example of failure to credit people in the correct way. [00:15:00] But it was definitely by accident and not by intent to exclude them. But it’s a sadness to me. Anyway this is the beginning of that story of three samples. Male of 54 years old and a female of 42. In parentheses it says dark. Meaning dark-skinned. And [name redacted] who had a duodenal ulcer and was light-skinned. But Jim Engle and — Ralph Engle and Jim Pert had seen something funny about these samples and sent them to me to look at. And I eventually was able to show that it was a difference in the beta-globin. Later again transferrin. [00:16:00] Some comments on the migration. Not very important. Much more important is the sample being the — there’s a comment. Looks like probably not exactly a 2a when the hemoglobin is present. And that turned out to be a difference in the haptoglobin allele carried by many African American individuals. That was reduced expression of the haptoglobin 2 gene so that the heterozygote was a little bit different. The 2a heterozygote was a little bit different. I think we called it 2a modified eventually. [00:17:00] I forget. But so that was what was being observed here. And comment that they’re the same with respect to haptoglobin but definitely a new group. September 26th. Family again and then on September 27th Thursday. Checked on the hemoglobin pattern versus 2a plus hemoglobin on these samples. With a rather full description of what was happening on the next — on the page preceding. And with a comment. [00:18:00] Two pages later. After September 27th. Evidence that the Negro serum contains some 1 characteristics as well as some of 2a is apparent from this result. So that they have some characteristics like type 1 and like 2a. But less than — that’s something else, not important. More comments. Haptoglobin 2a-1 [00:19:00] similar to but not exactly equal to 2-1. As I was saying. Still thinking mainly about the haptoglobin of these samples. This would suggest a possible [HV2?] something gene as well. A modified gene, 2a gene. And haven’t yet seen the beta-globin difference I think at this point. Friday September 26th an [ordinary?] family. And some more tests on September 28th of the [Jones?] and [World?] sample. [00:20:00] From the African Americans. So I have what I’m now calling 2a-1 compared to the usual 2-1. Now back to trying to get better first dimension by blotting fractionated starch in — starch grains that is with filter paper. Several pages of this not really very important. It never worked out. But September 30th is an example. October 2nd, etc. Continuing [00:21:00] October 3rd. Very promising indeed. I liked what I was blotting. I never did use it eventually. October 3rd Wednesday just some ordinary photography to demonstrate different sample. Using Type A film Kodachrome film. Notice on the left-hand side of this page there are comments of Negroes plus or minus hemoglobin versus David A. Scott DAS and cattle groups. I hadn’t yet gotten any interest in the book that I can see of the cattle groups. But I had begun to do samples from [00:22:00] that Charlie Hickman sent me from a different place in Canada, I just forget where for the moment, to look at. And they were very interesting, the cattle group. October 4th received from New York Hospital some more samples. [name redacted] something. Can’t read it. And [name redacted]. Probably a good deal of these four were lost in the mail, it says. Special delivery samples of unknown date from [name redacted], [name redacted], and [name redacted]. [00:23:00] With a comment. Eight days in the mail. Can’t take these very seriously. October 4th. Still trying blotting. On October 14th blotting starch grain electrophoresis. Sunday October 7th. A whole set of gels run by Otto. And Otto on October 9th. Tried some more photography on October 9th and October 14th. [00:24:00] So on October 10th latest batch of starch which was accidentally handling starch. Checking the various samples that I got from Negroes. 2a, 1, 2, 3, 4, 5, and some were what I called Negro 2a, which later was 2a-1. So there were two did types. October 11th. Family. Otto. So some samples again. From New York. They kept sending good samples. October 12th Friday. Mailed on the 10th. [00:25:00] And looks like Negro 2a as I called it. More from New York. So they were sending me many samples. And then I begin to understand. October 17th a whole bunch of samples were checked for 2a and all were found correct. And October 18th checked for 2b and 2a again. So making comments here on [00:26:00] things looking a little bit strange in the 2a samples. And hand comment following the — to repeat all the Negroes to date with 3% hemoglobin. And again some photography. Detail. With a cattle diagram. Commented on. But I don’t see the results on the cattle samples yet. But evidently we’d been doing cattle samples quite a lot. Or Charlie Hickman had been doing them. I’m not sure exactly what was going on here because I don’t have records of running many gels. I think he maybe ran the samples and sent me the results. Although there were — I have [00:27:00] received October 19th Friday five control bovine serum shipped on the 18th and four unknowns 1, 2, 3, and 4. And in pencil there beginning of the notation for the bovine samples is clear in Roman numerals in pencil IV, III, and IV, and II, and I. Except — although the separations were disappointingly fuzzy. But there is — evidently we already had a good hypothesis on the inheritance of these bands. Beta-globin bands. Because number one didn’t fit with our hypothesis. And I got a telegram from Charlie Hickman saying, [00:28:00] “Number one animal 5572 and two animal 55, etc., etc. are the progeny of whoever.” Something was a little bit weird. I know in the final paper that there’s some discussion of some you might say incorrectly recorded paternity of these artificially inseminated animals. Saturday October 10th some more comments on samples from cattle. Many [00:29:00] little odds and ends of comments on the results obtained on Tuesday October 28th of different samples. Trying to understand what was happening. Checked again Tuesday October 23rd. Free hemoglobin the same in all 21. And then haptoglobins were 2 — they were all 2b except for one individual that had no haptoglobin. Got some starch evidently from Ralph Engle Wednesday October 24th. [00:30:00] Ralph and Jim worked out a rather sophisticated method of understanding the starch by commercially available machine which measured the viscosity while you heated starch and was helpful in understanding how to hydrolyze the starch to get a reproducible product. And some of these Ralph Engle starches are commented on. October 24th. One and a half hour. One and three-quarters hours. Meaning the length of hydrolysis. And suggestion to the 13.8% is as strong as the regular starch. [00:31:00] But the gel shrinks rather a lot. Trace a granular comment on different starches. More samples from New York. On October 28th. October 25th some fairly substantial comments on the various types of sample obtained. Haptoglobin types obtained with Negro samples. Of course the normal 2b, etc., [name redacted] 2a, [00:32:00] [name redacted] 2b, etc., 1, 1, 1. Query 1, 1, 1, 2a, 2a. Notice there the 2 — the unusual 2a. More checks October 25th 1 and 2a haptoglobin all essentially normal. Here is the beginning of the understanding of the fact that there was a variant in the beta-globin, later transferrin, first became noticed on Friday October 26th where there’s a comment of [00:33:00] [name redacted] and [name redacted]. Abnormal. Query extra post beta. Looks as if I was beginning to realize that there was a heterozygous individual with two beta-globin, that is transferrin, bands. A diabetic family. October 20th — 27th. Some more comments on what samples are — what are comparing [name redacted], [name redacted], [name redacted]. Comparing [name redacted] with [name redacted] [00:34:00] and [name redacted] with [name redacted]. And it says that [name redacted] is definitely the same as [name redacted] but [name redacted] is not equivalent to [name redacted]. The extra zone is faster than the usual pace post beta. Two dimensions to locate it. So I decided to do a two-dimensional gel to try to find out what was happening. And next page is devoted to black-and-white photography. Need prints of mother shop twins etc. KB-17 was the film. Usual red filter, etc. Using the setup with a reflecting bulb. And 1-second exposure, 2, 4, 8, 16. At that time we used to — or shortly thereafter we used to take a whole series of exposures for every gel. Maybe four or five exposures. And then [00:35:00] print the best one. It was very laborious. And it ruined my enjoyment of photography because every day you had to develop these films and print them. And it became a terrible routine. So here the last entry in this book is therefore expose 1-second f/8, f/8 over 11, or f/11. Three different exposures for [mother shop?] samples and three sets of twins. And f/5.6 by half stops to f/16 for the latest bovine. So a whole bunch of images. The results very good. Probably f/11 is the closest after all to the last roll of film. [00:36:00] And a comment. The bovine samples require a higher contrast film. But that was the recording of the samples from cattle and from the Negroes. And that’s the end of physical book IX one X. [00:36:25]